Microtissue self-assembly is thought to be driven primarily by cadherins, while connexons have been examined mainly in intercellular coupling. We investigated whether connexon 43 (Cx43)-mediated cell adhesion modulates self-assembly of human KGN granulosa cells, normal human fibroblasts (NHFs), and MCF-7 breast cancer cells seeded into nonadhesive agarose gels. We found that treatment with anti-Cx43 E2 (112 mu g/ml), which suppresses Cx43 docking, significantly inhibited the kinetics of KGN and NHF self-assembly compared to the preimmune sera control (41.1 +/- 4.5 and 24.5 +/- 10.4% at 8 h, respectively). Likewise, gap junction inhibitor carbenoxolone also inhibited self-assembly of KGN, NHF, and MCF-7 cells in a dose-dependent manner that was specific to cell type. In contrast, Gap26 connexin mimetic peptide, which inhibits channel permeability but not docking, accelerated self-assembly of KGN and NHF microtissues. Experiments using selective enzymatic digestion of cell adhesion molecules and neutralizing N-cadherin antibodies further showed that self-assembly was comparably disrupted by inhibiting connexin-and cadherin-mediated adhesion. These findings demonstrate that connexon-mediated cell adhesion and intercellular communication differentially influence microtissue self-assembly, and that their contributions are comparable to those of cadherins.-Bao, B., Jiang, J., Yanase, T., Nishi, Y., Morgan, J. R. Connexon-mediated cell adhesion drives microtissue self-assembly. FASEB J. 25, 255-264 (2011). www.fasebj.org